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A high throughput beta-globin genotyping method by multiplexed melting temperature analysis.

Zhili Lin·Joseph G Suzow·Jamie M Fontaine·Edwin W Naylor
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Abstract

For a population-based newborn screening program, challenges exist in using technological advances to improve the quality and efficiency of the existing screening program and to develop new diagnostic capabilities. A newly developed genotyping method for screening of common mutations within the beta-globin gene is described here. This genotyping system consists of three major components: an automation system for high throughput DNA extraction and PCR setup, a conventional thermal cycler, and a LightTyper instrument for post-PCR melting temperature analysis. Briefly, genomic DNA is extracted from dried blood on a filter paper using methanol and Tris buffer. Genetic fragments of interest are amplified by asymmetric PCR. Fluorescent labeled probes are added during PCR setup, which eliminates the need for any post-PCR sample handling process. Melting temperature analysis is achieved through fluorescent resonance energy transfer (FRET) reaction using the LightTyper instrument. The assay is designed to simultaneously detect three common beta-globin mutations, S(A173T), C(G172A), and E(G232A), and can identify any of the eight possible genotypes in a single reaction: AA, AE, EE, AS, SC, SS, AC, and CC (A represents wild type allele). The method was validated with a large number of samples in both a retrospective and parallel study. Results were compared to those obtained by isoelectric focusing electrophoresis. The accuracy of this genotyping method is greater than 99%.

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DNAFluorescence Resonance Energy TransferGenotypeGlobinsHumansInfant, NewbornMutationPolymerase Chain ReactionTransition TemperatureJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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Zhili Lin, Joseph G Suzow, Jamie M Fontaine, Edwin W Naylor. 2004. A high throughput beta-globin genotyping method by multiplexed melting temperature analysis.. https://doi.org/10.1016/j.ymgme.2003.12.007

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